Javier Sanz-Zamora

Hyperlipidemia-associated atherosclerosis promotes plaque formation and extracellular matrix accumulation in the vascular media, increasing also the production of reactive oxygen species (ROS), inflammation, and the oxidation of LDL to oxidized-LDL, through mechanisms that remain unknown. Our objective was to determine whether oxidizedLDL-induced oxidative stress drives fibrosis in the arterial media during atherosclerosis and whether antioxidants can prevent or reduce this effect. Experiments were conducted in vascular smooth muscle cells (VSMCs) and in apolipoproteinE-knockout (KO-apoE) mice. ROS production was measured by confocal microscopy and fibrosis by immunofluorescence, Western and RT-qPCR in VSMCs and by Sirius red staining and RT-qPCR in mice. In VSMCs, oxidized-LDL treatment significantly increased the expression of TGF-beta, fibronectin and collagen-I, indicating fibrosis. Additionally, oxidized-LDL enhanced ROS production and NF-κB activation. The fibrotic response was prevented by treatment with the antioxidant N-acetylcysteine (NAC) and an NF-κB inhibitor, suggesting that the ROS-NF-κB axis mediates these effects in VSMCs. In vivo, KO-apoE mice exhibited higher cholesterol levels and increased ROS production compared with wild-type controls. These mice also showed enhanced aortic fibrosis. Importantly, NAC administration reduced both ROS production and fibrosis in KO-apoE mice. Overall, the findings suggest that both oxidized-LDL-induced vascular fibrosis and hyperlipidemia-induced fibrosis are mediated by ROS.

Age-related hyperphosphatemia is increasingly recognized as a contributing factor in sarcopenia. This work studies the metabolic effects of elevated phosphate on muscle. C2C12 cells were differentiated in the absence or presence of 10 mM β-glycerophosphate (BGP), an exogenous phosphate donor. In addition, quadriceps muscles from four experimental groups of male C57BL/6J mice were analyzed: young (5 months) and old (24 months) fed with standard diet; old mice fed with hypophosphatemic diet or supplemented with the phosphate binder Velphoro^®, for the last three months of life. Mice were stratified according to sarcopenia degree based on muscle mass, strength and physical performance. Protein levels were determined by immunoblotting and mRNA expression by RT-qPCR. ATP levels were measured by luminescence and L-lactate production, citrate synthase and cytochrome c oxidase activities by colorimetric assays. Mitochondrial content, membrane potential and reactive oxygen species (ROS) were determined by fluorescence assay. BGP-treated cells showed increased glucose transporter 1 (GLUT1) and decreased NADH Dehydrogenase (CI-NDUFB8) protein expression, elevated hexokinase II (HK2), phosphoglycerate kinase 1 (PGK1) and lactate dehydrogenase A (LDHA) mRNA levels, reduced ATP levels, increased lactate production, and decreased mitochondrial enzyme activities. Moreover, BGP increased ROS, diminished mitochondrial membrane potential, and altered fusion–fission dynamics and mitophagy. In aged quadriceps, oxidative phosphorylation (OXPHOS) subunits and superoxide dismutase 2 (SOD2) expression were reduced. The hypophosphatemic diet improved all parameters, whereas Velphoro^® selectively increased Mitochondrial cytochrome C oxidase subunit 1 (CIV-MTCO1) expression. Several altered mitochondrial markers are associated with sarcopenia degree. Altogether, hyperphosphatemia induces metabolic changes that scale with the sarcopenic degree. Our findings show a relevant association between hyperphosphatemia and mitochondrial dysfunction, and they support the potential benefit of phosphate reduction as a strategy to prevent or mitigate sarcopenia.